Butyrly-HIST1H3A (K27) Antibody

What is histone H3K27 butyrylation and how does it differ from other acylations?

Histone H3K27 butyrylation (H3K27bu) is a post-translational modification where a butyryl group is added to lysine 27 on histone H3. Unlike acetylation (two-carbon chain), butyrylation contains a four-carbon chain, creating structurally distinct modifications. This structural difference affects recognition by reader proteins - while bromodomains typically recognize acetylation, YEATS and double PHD finger domains better accommodate longer acyl chains like butyrylation . Mass spectrometry studies have demonstrated that H3K27bu produces a lower signal than H3K27ac at similar abundances, suggesting butyrylation may be underestimated in many studies without synthetic standards for quantification .

Which tissues show significant levels of histone butyrylation and why?

The cecum and distal intestine exhibit particularly high levels of histone butyrylation compared to other tissues . This tissue-specific distribution correlates with these regions being major sites of microbial fermentation and production of short-chain fatty acids (SCFAs) including butyrate . Mass spectrometry analysis has specifically identified butyrylation at lysines 9 and 27 on histone H3 (H3K9bu and H3K27bu) in cecum samples . The localized abundance in intestinal epithelial cells suggests these modifications play a role in gut-specific gene regulation and may represent a mechanism through which the microbiome influences host gene expression through chromatin modification .

How is histone butyrylation regulated in vivo?

Histone butyrylation is primarily regulated through two key mechanisms: microbiota-dependent production of short-chain fatty acids and metabolic availability of butyryl-CoA. Studies have demonstrated that intestinal epithelial cells from germ-free or antibiotic-treated mice show reduced H3K27bu levels compared to conventionally raised animals . Additionally, supplementation with tributyrin (a butyrate prodrug) can rescue histone butyrylation levels in microbiota-depleted mice, confirming the direct metabolic link between butyrate availability and histone butyrylation . The enzymatic regulation of histone butyrylation involves writers that add the modification, erasers that remove it, and readers that recognize the mark, though these specific enzymes for butyrylation remain less characterized than those for acetylation .

How specific are commercial H3K27bu antibodies and what cross-reactivities should researchers be aware of?

Commercial antibodies targeting H3K27bu demonstrate varying degrees of specificity. Testing has shown that antibodies targeting H3K27bu show some cross-reactivity with H3K9bu and crotonylation, but generally minimal cross-reactivity with H3K27ac . Conversely, antibodies targeting H3K27pr (propionylation) may exhibit substantial cross-reactivity with H3K27bu . When selecting an antibody for H3K27bu research, researchers should review validation data specifically testing against closely related modifications. For example, ab241464 antibody has been validated for specificity using recombinant nucleosomes with defined modifications, showing selectivity for the butyryl modification .

What validation steps are essential before using an H3K27bu antibody for experiments?

Before employing an H3K27bu antibody in critical experiments, researchers should perform comprehensive validation:

• Peptide competition assays to confirm specificity for the butyrylated epitope

• Dot blot analysis against a panel of modified peptides (H3K27ac, H3K27bu, H3K9bu, H3K27cr)

• Western blot validation comparing butyrylation-enhanced samples (e.g., sodium butyrate-treated cells) versus controls

• Testing with recombinant nucleosomes containing specific modifications

• Immunoprecipitation followed by mass spectrometry to confirm target identity

Testing has shown that when validating with recombinant nucleosomes, H3K27bu antibodies should demonstrate selectivity toward butyrylation with minimal cross-reactivity to acetylation . Additionally, validation in cellular contexts should include positive controls such as intestinal tissue samples or cells treated with butyrate to enhance the modification .

How can researchers troubleshoot weak or non-specific signals when using H3K27bu antibodies?

When experiencing weak or non-specific signals with H3K27bu antibodies, researchers should consider several optimization strategies:

• Increase antibody concentration, as H3K27bu may be less abundant than other modifications like H3K27ac

• Enhance butyrylation levels in positive controls using sodium butyrate treatment (typically 30mM for 4 hours)

• Modify blocking conditions to reduce background (test different blockers like BSA, milk, or commercial blocking reagents)

• Increase sample amount, as mass spectrometry suggests butyryl marks become undetectable at levels where acetyl marks remain visible

• Optimize detection methods with more sensitive systems like HRP-conjugated secondary antibodies with enhanced chemiluminescence

For western blotting specifically, researchers have successfully detected H3K27bu using 1/2000 dilution of antibody on lysates from cells treated with sodium butyrate . For immunocytochemistry, more concentrated antibody (1/30 dilution) may be required along with signal amplification systems .

What are the optimal protocols for ChIP-seq using H3K27bu antibodies?

Optimizing ChIP-seq for H3K27bu requires several specific considerations:

• Crosslinking: Standard 1% formaldehyde for 10-15 minutes is typically sufficient, but more extensive crosslinking may help capture transient interactions.

• Chromatin shearing: Aim for fragments of 200-500bp for optimal resolution.

• Antibody amount: As H3K27bu may be less abundant than H3K27ac, using 2-5µg of antibody per ChIP reaction is recommended.

• Washing conditions: Include high-salt washes to minimize cross-reactivity with other acylations.

• Controls: Include input chromatin, IgG negative controls, and H3K27ac ChIP performed in parallel for comparison.

• Library preparation: Standard ChIP-seq library preparation protocols are applicable.

• Bioinformatic analysis: Be aware that H3K27bu peaks may overlap with but differ from H3K27ac peaks, and may be enriched at genes involved in oxidative stress response pathways .

Researchers should note that according to research findings, H3K27bu is associated with active gene regulatory elements and correlates with gene expression levels, suggesting peak distribution patterns similar to active marks like H3K27ac .

How should researchers prepare intestinal tissue samples for H3K27bu detection?

For optimal H3K27bu detection in intestinal tissues, researchers should follow these specific preparation steps:

• Sample collection:

  • For mouse models, carefully collect the cecum and distal intestine, which show highest levels of histone butyrylation .

  • Process samples rapidly to prevent degradation of the modification.

• Epithelial cell isolation:

  • Perform EDTA-based epithelial stripping to isolate intestinal epithelial cells, where H3K27bu is primarily observed.

  • Alternatively, use gentle mechanical separation of epithelium from underlying tissue.

• For immunohistochemistry/immunofluorescence:

  • Fix tissues in 4% formaldehyde (10-15 minutes) .

  • Permeabilize using 0.2% Triton X-100 and block with 10% normal serum .

  • Incubate with primary antibody at appropriate dilution (commonly 1/30 for H3K27bu) .

• For histone extraction:

  • Homogenize tissue in lysis buffer containing histone deacetylase inhibitors.

  • Extract histones using acid extraction methods.

  • For optimal results, process samples immediately or flash-freeze in liquid nitrogen.

• For enhanced butyrylation detection:

  • Consider butyrylation-enhancing treatments like tributyrin administration to increase signal strength .

What mass spectrometry approaches are most suitable for analyzing H3K27bu?

Mass spectrometry analysis of H3K27bu requires specific methodological considerations:

• Sample preparation:

  • Extract histones using acid extraction from tissues or cells.

  • Chemical derivatization of unmodified lysines improves identification of modified residues.

  • Enzymatic digestion with trypsin following propionylation of unmodified lysines.

• Analytical approaches:

  • Liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) has successfully identified H3K27bu and H3K9bu in cecum samples .

  • Tandem MS (MS/MS) is essential for assigning modifications to specific lysines in peptides with multiple potential modification sites .

• Quantification strategies:

  • Use synthetic standards for accurate quantification, as H3K27bu produces lower signal than H3K27ac at similar abundances .

  • Consider dilution series of acylated nucleosomes spiked into unmodified nucleosomes to establish detection limits.

• Data analysis considerations:

  • Search for butyryl (+70 Da) modifications on lysine residues.

  • Be aware that butyryl marks become undetectable at levels where acetyl marks remain visible, suggesting potential underestimation .

  • Analyze histone H3 peptides 9-17, 18-26, and 27-40 for comprehensive modification profiling .

Mass spectrometry analysis has revealed that the 27-40 peptide displays complex modification patterns with butyrylation on K27 often existing alongside other modifications .

How can researchers investigate the relationship between microbiota, tributyrin, and H3K27bu levels?

To investigate the relationship between microbiota, tributyrin, and H3K27bu levels, researchers can implement these methodological approaches:

• Experimental models:

  • Compare conventional vs. germ-free or antibiotic-treated mice to assess microbiota dependence .

  • Administer tributyrin (a butyrate prodrug) via oral gavage or dietary supplementation to rescue H3K27bu levels in microbiota-depleted mice .

  • Use defined bacterial communities with different SCFA production capabilities.

• Analytical techniques:

  • Western blot analysis of H3K27bu normalized to total H3 levels in intestinal epithelial cells.

  • ChIP-seq to identify genomic regions where H3K27bu is affected by microbiota depletion or tributyrin supplementation.

  • RNA-seq in parallel to correlate gene expression changes with H3K27bu alterations.

  • Measure SCFA concentrations in intestinal contents using GC-MS to correlate with H3K27bu levels.

• Data integration:

  • Correlate microbiome composition (16S rRNA sequencing) with H3K27bu levels.

  • Perform pathway analysis of genes associated with H3K27bu in different conditions.

  • Compare H3K27bu genomic distribution patterns between conventional, antibiotic-treated, and tributyrin-rescued models.

Research has demonstrated that tributyrin treatment rescues gene expression changes in microbiota-depleted models, with RNA-seq analysis showing that most genes rescued by tributyrin treatment are downregulated .

What approaches can distinguish direct effects of H3K27bu from other histone modifications in gene regulation?

Distinguishing the direct effects of H3K27bu from other histone modifications requires sophisticated experimental approaches:

• Genomic approaches:

  • Perform comparative ChIP-seq for multiple histone marks (H3K27bu, H3K27ac, H3K27me3, etc.) to identify regions uniquely enriched for H3K27bu.

  • Apply sequential ChIP (re-ChIP) to determine co-occurrence or mutual exclusivity of H3K27bu with other modifications.

  • Integrate with ATAC-seq or DNase-seq to correlate with chromatin accessibility changes.

• Functional genomics:

  • Use CRISPR-based approaches to target writers/erasers of butyrylation.

  • Apply nascent RNA sequencing to detect immediate transcriptional responses to changes in H3K27bu levels.

  • Perform time-course analyses following manipulation of H3K27bu to separate direct from indirect effects.

• Biochemical strategies:

  • Develop in vitro transcription systems using reconstituted chromatin with defined H3K27bu modifications.

  • Identify and characterize proteins that specifically recognize H3K27bu (readers).

  • Conduct structural studies of H3K27bu interactions with nuclear proteins.

• Data analysis:

  • Apply multivariate analysis to distinguish effects of different histone marks.

  • Develop computational models incorporating multiple datasets to predict functional outcomes.

Current research suggests that H3K27bu is associated with active gene regulatory elements, but determining its unique contributions remains challenging, with evidence that most tributyrin-rescued genes (likely regulated by H3K27bu) are downregulated, contrasting with the generally activating role attributed to acetylation .

What is the current understanding of H3K27bu's role in oxidative stress response in intestinal epithelial cells?

The relationship between H3K27bu and oxidative stress response in intestinal epithelial cells represents an emerging area of research:

• Genomic associations:

  • ChIP-seq analysis has revealed that top peaks of H3K27bu in mouse cecal epithelial cells are enriched in genes involved in oxidative stress and cellular adaptations .

  • H3K27bu levels correlate with gene expression, suggesting functional importance in regulating stress response genes .

• Metabolic connections:

  • Butyrate and tributyrin have been reported to reduce oxidative stress in multiple studies .

  • This creates a potential feedback mechanism where the same metabolites that induce H3K27bu also modulate oxidative stress pathways.

• Microbial influence:

  • Microbiota depletion affects both H3K27bu levels and expression of oxidative stress response genes.

  • This suggests a mechanism by which disruption of the microbiome could alter epithelial stress responses through changes in histone butyrylation.

• Research status:

  • The causal relationship between H3K27bu and stress response gene regulation remains under investigation.

  • Studies have observed that histone butyrylation potentially has a functional role in cellular response to stress, but additional mechanistic studies are needed .

  • Whether H3K27bu specifically promotes stress resilience or is induced as part of the stress response remains unclear.

Future studies are needed to delineate the exact mechanisms connecting H3K27bu to oxidative stress response, including identification of specific transcription factors and co-regulators involved in this process .

How do H3K27bu distribution patterns compare between different tissue types?

Analysis of H3K27bu distribution patterns across tissues reveals significant tissue-specific variations:

• Tissue abundance patterns:

  • The cecum and large intestine display dramatically higher levels of histone butyrylation compared to other tissues .

  • This correlates with these regions being major sites of microbial fermentation and SCFA production .

  • Other tissues show minimal H3K27bu under normal physiological conditions.

• Cellular localization:

  • Within intestinal tissue, H3K27bu is primarily detected in epithelial cells rather than lamina propria or muscular layers.

  • Nuclear localization can be visualized using immunofluorescence microscopy, with highest signal intensity in actively dividing crypt cells .

• Genomic distribution:

  • In intestinal epithelial cells, H3K27bu is associated with active gene regulatory elements .

  • ChIP-seq analysis reveals enrichment at genes involved in oxidative stress response and cellular adaptation .

  • This distribution pattern differs from that observed in cultured cell lines treated with exogenous butyrate.

• Comparison with other modifications:

  • H3K27bu shows partial overlap with H3K27ac distribution, suggesting both shared and distinct regulatory functions.

  • Unlike the broad distribution of H3K27ac across many tissues, significant H3K27bu is primarily restricted to the intestinal environment.

These tissue-specific distribution patterns highlight the importance of studying histone butyrylation in physiologically relevant contexts rather than relying solely on cell culture models with exogenous butyrate treatment.

What are the major challenges in interpreting H3K27bu ChIP-seq data?

Interpreting H3K27bu ChIP-seq data presents several specific challenges:

• Antibody specificity concerns:

  • Cross-reactivity with other acylations, particularly H3K9bu and crotonylation .

  • Variable specificity between antibody batches and manufacturers requires validation controls.

  • Background signal interpretation can be complicated by partial cross-reactivity.

• Signal detection limitations:

  • H3K27bu may be less abundant than H3K27ac, affecting peak calling sensitivity .

  • Mass spectrometry suggests butyryl marks may be underdetected, potentially leading to underestimation of true modification distribution .

• Biological complexities:

  • H3K27bu levels are significantly influenced by microbiota and diet, creating potential batch effects .

  • The modification shows high tissue specificity, limiting the utility of standard control cell lines.

  • Different cell types within intestinal epithelium may have varying H3K27bu patterns.

• Data integration challenges:

  • Determining causality between H3K27bu presence and gene expression changes.

  • Distinguishing direct effects from indirect consequences of altered cellular metabolism.

  • Separating H3K27bu-specific effects from those of other simultaneously occurring modifications.

• Functional interpretation:

  • Research indicates H3K27bu is associated with both up and down-regulated genes, complicating functional assignment .

  • Determining whether H3K27bu is causative or consequential remains particularly challenging.

These challenges necessitate careful experimental design with appropriate controls, validation steps, and integration of multiple data types for accurate interpretation of H3K27bu ChIP-seq data.

How can researchers quantitatively compare H3K27bu levels across different experimental conditions?

For quantitative comparison of H3K27bu levels across experimental conditions, researchers should employ these methodological approaches:

• Western blot quantification:

  • Normalize H3K27bu signal to total H3 to control for histone extraction efficiency.

  • Include recombinant modified histones as standards for calibration.

  • Apply digital imaging and densitometry with appropriate statistical analysis.

  • Compare samples processed simultaneously to minimize technical variation.

• Mass spectrometry-based approaches:

  • Use isotopically labeled synthetic peptides with butyryl modifications as internal standards.

  • Account for lower sensitivity of butyryl detection compared to acetyl modifications .

  • Apply selected reaction monitoring (SRM) for targeted quantification.

  • Calculate modification stoichiometry relative to unmodified peptides.

• ChIP-qPCR analysis:

  • Design primers targeting regions with known H3K27bu enrichment.

  • Normalize to input DNA and include invariant genomic regions as controls.

  • Use spike-in chromatin for between-sample normalization.

  • Apply percent-of-input or fold-enrichment calculations consistently.

• ChIP-seq comparative methods:

  • Use consistent peak calling parameters across all datasets.

  • Apply appropriate normalization methods (spike-in, quantile normalization).

  • Compare both peak intensity and distribution patterns.

  • Utilize differential binding analysis tools with appropriate statistical models.

When comparing different modifications (e.g., H3K27bu vs. H3K27ac), researchers should account for the observation that H3K27bu produces lower signals than H3K27ac at similar abundance levels, potentially leading to underestimation of butyrylation .

Table 1: Comparison of Histone H3K27 Modifications

Table 2: Methodological Approaches for Studying H3K27bu